Metabolic adaptations of Mycobacterium tuberculosis (Mtb) to its host have become a focus of recent research, yet a comprehensive understanding of Mtb's nutrient requirements and metabolic pathways necessary for growth and persistence within its host is lacking. The majority of genes encoding enzymes in central carbon metabolism are required for growth of Mtb and, thus, belong to the class of in vitro essential genes, which are viewed as potential new drug targets. Mtb kills approximately 5000 people every day and new drugs are urgently needed to limit the impact of tuberculosis on global public health. Significant evidence suggests that Mtb metabolizes primarily fatty acids and synthesizes essential carbohydrates via gluconeogenesis during growth and persistence in vivo. However our preliminary data suggest that both glycolysis and gluconeogenesis are required for virulence, especially during chronic Mtb infections. A goal of this proposal is to determine the importance of every gene involved in glycolysis and gluconeogenesis for growth and persistence of Mtb in vitro, in macrophages and in mice. We will test the hypotheses that the stage of the infection and the immune-status of the host can control which carbon source is available to Mtb. We will measure Mtb's vulnerability to incomplete inhibition of enzymes involved in glycolysis and/or gluconeogenesis. Our work will provide a generally applicable blue print for the systematic evaluation of metabolic enzymes and pathways in vitro and in vivo and identify the glycolytic and gluconeogenic enzymes most attractive for drug development. PUBLIC HEALTH RELEVANCE: Tuberculosis is one of the world's most devastating diseases. It is responsible for more than two million deaths and eight million new cases annually. Work outlined in this proposal will investigate the importance of two pathways in central carbon metabolism for virulence of Mycobacterium tuberculosis. It will evaluate metabolic enzymes and pathways in vitro and in vivo and identify the glycolytic and gluconeogenic enzymes that are most attractive for tuberculosis drug development.